PROJECT SUMMARY Due to the increasing prevalence of obesity in the world, understanding the mechanisms involved in adipocyte metabolism and the conditions that lead to insulin resistance and diabetes is important. In adipose tissue lipids are stored as triglyceride (TAG) within lipid droplets (LD) that are tightly regulated by an assortment of proteins. When the body needs energy, TAG from the LD is hydrolyzed to release free fatty acids (FFA) in a process called lipolysis. In fasting states, circulating FFAs are utilized by distal tissues for energy, whereas in brown adipocytes lipolysis provides fatty acids to be used for adaptive thermogenesis. In obese individuals, dysregulated lipolysis causes the release of an excess of FFA, which can lead to insulin resistance. Perilipin (plin1) is a protein found on the surface of LD that regulates lipolysis by acting as scaffold for many of the enzymes and adapter proteins involved with hydrolyzing TAG into FFA. The goal of this grant is to provide training for the applicant in order to study how activation of cyclic-AMP dependent protein kinase (PKA) regulates plin1 activity and the mechanisms by which plin1 recruits the lipolytic machinery to the LD surface, as well as its role in systemic metabolism. Specifically, our preliminary results show that a family of proteins called the 14-3-3 proteins associate with plin1 at a novel phosphorylation site that we hypothesize is a critical regulator of stimulated-lipolysis. We will study the role of 14-3-3 proteins binding to plin1 in regulating PKA- stimulated lipolysis using two different experimental approaches: 1) mutating the binding site within plin1 and 2) by expressing a dominant negative form of the 14-3-3 proteins, thereby sequestering the 14-3-3 proteins, within cultured adipocytes. We will determine the specific proteins that are associated with plin1 during lipolysis as well as the changes in proteins that are bound after mutation of the 14-3-3 binding site within plin1 by using mass spectrometry based proteomics. As confirmation of the proteomics work, we will investigate direct interactions of the various proteins bound to plin1 using confocal microscopy and fluorescence resonance energy transfer. Finally, we will generate a mouse that has a mutation in the 14-3-3 binding site within plin1 in vivo using CRISPR-mediated gene editing. These mice will be used to evaluate the effects of the mutation on changes in body composition, brown adipocyte thermogenesis, insulin-glucose homeostasis, adipocyte physiology including lipolysis, and the lipolytic complex of proteins that associate with plin1 to regulate lipolysis.